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Xenobiotica
the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Cross-species comparison of the metabolism and
excretion of selexipag
Tomohiko Ichikawa, Tetsuhiro Yamada, Alexander Treiber, Carmela Gnerre,
Jérôme Segrestaa, Swen Seeland & Kiyoko Nonaka
To cite this article: Tomohiko Ichikawa, Tetsuhiro Yamada, Alexander Treiber, Carmela Gnerre,
Jérôme Segrestaa, Swen Seeland & Kiyoko Nonaka (2018): Cross-species comparison of the
metabolism and excretion of selexipag, Xenobiotica, DOI: 10.1080/00498254.2018.1444814
To link to this article: https://doi.org/10.1080/00498254.2018.1444814
Accepted author version posted online: 22
Feb 2018.
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Cross-species comparison of the metabolism and excretion of selexipag
Tomohiko Ichikawa1, Tetsuhiro Yamada1, Alexander Treiber2, Carmela Gnerre2, Jérôme
Segrestaa2, Swen Seeland2, Kiyoko Nonaka1
1Pharmacokinetics and Safety Assessment Department, Nippon Shinyaku Co., Ltd, Kyoto, Japan,
and 2Preclinical Pharmacokinetics and Metabolism, Actelion Pharmaceuticals Ltd, Allschwil,
Switzerland
Address for correspondence: Tomohiko Ichikawa, Pharmacokinetics and Safety Assessment
Department, Nippon shinyaku Co., Ltd, 14, Nishinosho-monguchi-cho, Kisshoin, Minami-ku, Kyoto
601-8550, Japan. Tel: +81 75 321 9113. Fax: +81 75 314 3269. Email:
to.ichikawa@po.nippon-shinyaku.co.jp
Abstract
1. The metabolism of the prostacyclin receptor agonist selexipag (NS-304; ACT-293987) and its
active metabolite MRE-269 (ACT-333679) has been investigated in liver microsomes and
hepatocytes of rats, dogs, and monkeys. MRE-269 formation is the main pathway of selexipag
metabolism, irrespective of species. Some interspecies differences were evident for both
compounds in terms of both metabolic turnover and metabolic profiles. The metabolism of
MRE-269 was slower than that of selexipag in all three species.
2. The metabolism of selexipag was also studied in bile-duct-cannulated rats and dogs after a
single oral and intravenous dose of [14C]selexipag. MRE-269 acyl glucuronide was found in
both rat and dog bile. Internal acyl migration reactions of MRE-269 glucuronide were identified
in an experiment with the synthetic standard MRE-6001.
3. MRE-269 was the major component in the faeces of rats and dogs. In ex vivo study using rat and
dog faeces, selexipag hydrolysis to MRE-269 by the intestinal microflora is considered to be a
contributory factor in rats and dogs.
4. A taurine conjugate of MRE-269 was identified in rat bile sample. Overall, selexipag was
eliminated via multiple routes in animals, including hydrolysis, oxidative metabolism,
conjugation, intestinal deconjugation, and gut flora metabolism.
Keywords
Prostacyclin receptor agonist, selexipag, metabolism, species difference, rat, dog, monkey
Accepted Manuscript
Introduction
Selexipag
(2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide) is a
potent, orally active, selective prostacyclin receptor agonist which is rapidly hydrolysed to its active
metabolite MRE-269 ({4-[(5,6-diphenylpyrazin-2-yl)(propan-2-yl)amino]butoxy}acetic acid)
(Kuwano et al., 2007; Asaki et al., 2015).
In the clinic, selexipag demonstrated a significant improvement in disease progression in patients
with pulmonary arterial hypertension (PAH) (Scott, 2016). The US Food and Drug Administration
approved selexipag for the treatment of PAH in 2015, while the European Medicines Agency and the
Japanese Pharmaceuticals and Medical Devices Agency approved selexipag for the treatment of
PAH in 2016.
Pharmacokinetic studies of selexipag in rats, dogs and monkeys have been reported previously
(Ichikawa, 2018). These studies demonstrate that selexipag is absorbed rapidly after oral dosing (the
absolute bioavailability is 27% in monkeys), displays a low volume of distribution in monkeys and
is excreted in the faeces of all three species, with a smaller fraction of radioactivity excreted in the
urine. In a study with healthy volunteers, selexipag exhibited good oral bioavailability (49%) and
displayed a low volume of distribution of approximately 12 L (Kaufmann, 2017). Excretion studies
in Sprague-Dawley rats, beagle dogs and cynomolgus monkeys after administration of
[14C]selexipag reveal that a major portion of the radioactivity is recovered in the faeces (Ichikawa,
2018). Studies with bile-duct-cannulated rats and dogs dosed orally with [14C]selexipag have
demonstrated that bile is the primary route of excretion, because at least 66% of the dose is
recovered in the bile, whereas only 5.5% of the dose is recovered in the urine of bile-duct-cannulated
animals. In the human absorption, distribution, metabolism, and excretion study with [14C]selexipag,
faecal recovery accounted for 93% of the administered dose, whereas urinary recovery accounted for
only 12% (Kaufmann, 2012), suggesting that renal elimination is not as important in humans as it is
in animals (Ichikawa, 2018). Gnerre et al (2017) have recently reported the pathways of selexipag
metabolism in vivo in humans. Although the routes of elimination of selexipag in rats, dogs and
monkeys have been determined, the metabolic pathways in these species have not been fully
characterised.
The present study was conducted to evaluate the comparative metabolic disposition of selexipag
in different species. The study included both an in vitro component, in which liver microsomes and
hepatocytes of rats, dogs, and monkeys were incubated with [14C]selexipag, and an in vivo
component, in which rats and dogs were dosed orally or intravenously with [14C]selexipag and
plasma, urine, faeces, and bile were collected and analysed for radioactive components. In addition,
the elimination properties of [14C]selexipag and its active metabolite [14C]MRE-269 were studied in
bile-duct-cannulated rats and dogs after oral or intravenous administration of [14C]selexipag.
Accepted Manuscript
Metabolic profiles were recorded using high performance liquid chromatography (HPLC) coupled to
a radiodetector and metabolites structurally identified using MS and authentic reference compounds.
Materials and Methods
Chemicals and Reagents
[14C]Selexipag and its metabolite [14C]MRE-269 (Figure 1) were synthesised from
[ring-U-14C]benzil at Nippon Shinyaku. The specific activity of the [14C]selexipag was 4.15 MBq
(112 μCi) per mg and that of the [14C]MRE-269 was 5.00 MBq (135 μCi) per mg. The radiochemical
purity of both compounds was 98%. Unlabelled selexipag and the metabolite standards MRE-269
and MRE-6001 were synthesised at Nippon Shinyaku. β-glucuronidase was purchased from Roche
Diagnostics GmbH (Penzberg, Germany), glucose-6-phosphate (disodium salt) and NADP from
Sigma (Buchs, Switzerland) and glucose-6-phosphate dehydrogenase from Boehringer Mannheim
(Rotkreuz, Switzerland). General purpose reagents and solvents were of analytical grade (or a
suitable alternative) and were obtained principally from Nacalai Tesque (Kyoto, Japan) and Wako
chemicals (Osaka, Japan). The liquid scintillation cocktails used were Emulsifier Scintillator Plus,
Hionic-Fluor (PerkinElmer, Waltham, MA), Optiflow Safe 2 (Berthold Technologies GmbH,
Regensdorf, Switzerland) and Irga Safe plus (PerkinElmer, Zürich, Switzerland). Pooled liver
microsomes (20 mg/mL) of Sprague-Dawley rats, beagle dogs and cynomolgus monkeys were
purchased from Becton Dickinson (Franklin Lakes, NJ). Pooled intestinal microsomes (10 mg/mL)
of Sprague-Dawley rats and beagle dogs were purchased from XenoTech LLC (Lenexa, KS).
Cryopreserved beagle dog and cynomolgus monkey hepatocytes were purchased from Biopredic
International (Rennes, France) and hepatocytes from Sprague-Dawley rats by Celsis In Vitro
Technologies (Leipzig, Germany).
The study was performed at Nippon Shinyaku, Actelion Pharmaceuticals (Allschwil,
Switzerland) and Charles River Laboratories (Tranent, Edinburgh, UK). Protocols and procedures
involving animals were conducted in accordance with the Internal Regulations on Animal
Experiments at Nippon Shinyaku, which are based on the Law for the Humane Treatment and
Management of Animals (Law No. 105, 1 October 1973, as amended on 1 June 2006), the guidelines
of the Baselland Cantonal Veterinary Office, or the guidelines of the Institutional Animal Care and
Use Committee at Charles River, as appropriate.
NADPH-regenerating system
The NADPH-regenerating system used for the microsomal incubations was prepared as a 10-fold
concentrated stock solution and kept at –20 °C. It consisted of 11 mM NADP, 100 mM
glucose-6-phosphate and 50 mM magnesium chloride in 0.1 M phosphate buffer (pH 7.4).
Glucose-6-phosphate dehydrogenase (20 UI/mL) was added before use.
Accepted Manuscript
Incubation of selexipag and MRE-269 with liver microsomes and hepatocytes
For liver microsomes, the incubation mixture contained 3 mg/mL microsomal protein, 10 μM, 0.56
μCi/mL [14C]selexipag (diluted from a 2 mM, 100 μCi/mL stock solution in acetonitrile), 5 mM
MgCl2, and 100 mM potassium phosphate buffer (pH 7.4) in a total volume of 100 μL. Reactions
were initiated by the addition of the NADPH regenerating system (final concentration of NADP, 1
mM) and incubated at 37 °C in a shaking incubator block (450 rpm). After 60 min, reactions were
terminated with 100 μL of ice-cold methanol. Control incubations were performed without NADPH
or liver microsomes. Samples were then centrifuged at 20800×g and the supernatants subjected to
HPLC analysis. [14C]MRE-269 was incubated with liver microsomes using the same procedure.
The metabolism of [14C]selexipag was evaluated in rat, dog, and monkey hepatocytes.
Incubations were performed in modified Williams’ medium E fortified with 1.0 μM hydrocortisone
and 400 μM glutamine. The final substrate concentration in the cell suspension was 10 μM
(0.56 μCi/mL) in a volume of 0.2 mL at a cell density of 1 × 106 cells/mL. Incubation proceeded for
24 h at 37 °C in a 24-well plate in a humid atmosphere containing 5% CO2. To measure the recovery
of compound, a 5-μL aliquot of each incubation mixture was added to 4 mL of scintillation cocktail
before and after centrifugation of the incubation mixtures. Reactions were terminated with 200 μL of
methanol containing 0.1 M hydrochloric acid to prevent post-sample degradation of the putative
MRE-269 acyl glucuronide (Kaufmann et al., 2015). The supernatants were subjected to HPLC
analysis. [14C]MRE-269 was incubated with hepatocytes using the same procedure described for
[14C]selexipag.
Incubation of selexipag with intestinal microsomes
The metabolism of [14C]selexipag was evaluated in intestinal microsomes from Sprague-Dawley
rats and beagle dogs as described above for liver microsomes.
Sample collection
Rats
For the collection of plasma, [14C]selexipag was given orally to a naïve male Sprague-Dawley rat
(body weight, approximately 300 g; RCC, Füllinsdorf, Switzerland) at a dose level of 20 mg/kg
(200 μCi/kg). Blood samples were collected from the rat after oral dosing at a single time point of 30
min post-dose via cardiac puncture under isoflurane anaesthesia. For the collection of excrement,
male bile-duct-cannulated Sprague-Dawley rats (body weight, 233–317 g; RCC) received oral or
intravenous [14C]selexipag at dose levels of 20 mg/kg and 0.9 mg/kg, respectively, each containing
radioactive target doses of 100 μCi/kg. Prior to dosing, the animals underwent bile-duct-cannulation
surgery as described by Treiber et al. (2016). Bile, urine and faeces were collected at predefined
intervals over periods of 80 h for intravenous and 96 h for oral administration (Table 1).
Accepted Manuscript
Dogs
Bile-duct-cannulated dogs (body weight, approximately 13.5 kg) received oral and intravenous
[14C]selexipag at doses of 2 and 1 mg/kg, respectively, and intravenous [14C]MRE-269 at a dose of 1
mg/kg. The radioactive doses were 200 μCi/animal for both compounds. Prior to dosing, the animals
underwent bile-duct-cannulation surgery as described by Treiber et al. (2016). Urine and bile were
quantitatively collected into containers cooled by solid CO2 for the periods 0–6 and 6–24 h, and then
for 24-h periods up to 96 h post-dose. Faeces were quantitatively collected for 24-h periods up to 96
h post-dose. Whole-blood samples (ca 6 mL per time point) were collected from the jugular vein into
heparinised tubes at 0.083, 1, 2, 4, 8, 24, and 48 h post-dose.
Sample preparation for metabolic analysis
Rat plasma and urine samples
Blood (3 mL) was collected on ice into vials containing EDTA as anticoagulant at the single time
point of 30 min after oral dosing. Formic acid (0.5% of total blood volume) was added to prevent
degradation of putative phase 2 metabolites and plasma was prepared by centrifugation. Another
0.5% (v/v) formic acid was added and the acidified plasma sample stored at –20 °C pending analysis.
For the determination of total radioactivity, 5-μL aliquots of plasma were mixed with 4 mL of liquid
scintillation cocktail and analysed in a liquid scintillation counter (Tricarb 2300 TR; PerkinElmer).
For the recording of metabolic profiles, plasma was supplemented with three equivalents of an 8:2
(v/v) mixture of methanol and acetonitrile, acidified with 0.1% formic acid to precipitate protein,
homogenised on a Vortex mixer (Merck, Zürich, Switzerland) for about 30 s, and centrifuged at
20800×g for 10 min at 10 °C. The supernatant was evaporated to dryness using an EZ-2 Evaporator
(GeneVac Ltd, Ispwich, UK), reconstituted in 500 μL of a 1:1 (v/v) mixture of methanol and water
acidified with 0.1% formic acid and centrifuged at 20800×g for 5 min at 10 °C prior to HPLC
analysis. For identification of the MRE-269 acyl glucuronide by co-chromatography, a 1 mM stock
solution of the synthetic standard MRE-6001 was prepared in acidified methanol and 1.0-μL aliquots
of the stock solution were spiked into the plasma prior to sample work-up.
Urine was collected in metabolic cages over a period of 80 or 96 h. For the determination of total
radioactivity, 30-μL aliquots of urine were mixed with 4 mL of liquid scintillation cocktail and
analysed in a Tricarb 2300 TR liquid scintillation counter. For HPLC analysis, the urine samples
were centrifuged at 20800×g for 5 min at 10 °C and an aliquot of the supernatant was subjected to
analysis without further treatment.
Dog plasma and urine samples
Blood was collected from the jugular vein into heparinised tubes at predefined times over a period of
48 h after oral and intravenous dosing. One aliquot was retained for the analysis of total radioactivity
by liquid scintillation counting. Plasma was separated from the remaining sample by centrifugation.
Accepted Manuscript
For the recording of metabolic profiles, 1.5 mL of plasma was mixed with 4.5 mL of a mixture of
methanol/acetonitrile (8:2, v/v) acidified with 0.1% (v/v) formic acid, vortex-mixed for about 30 s
and then centrifuged at 20800×g for 10 min at 10 °C. The supernatant was evaporated to dryness in
an EZ-2 evaporator, the residue reconstituted in 500 μL of a 1:1 (v/v) mixture of methanol/water
acidified with 0.1% (v/v) formic acid, mixed for 5 min on a Vortex mixer and centrifuged at
20800×g for 10 min at 10 °C. The supernatants were subjected to HPLC analysis.
Urine was collected into containers over a period of 96 h. At the end of each collection period,
the urine was acidified with 1 M hydrochloric acid at a ratio of 9:1 (v/v). For the recording of
metabolic profiles, urine aliquots were centrifuged at 20800×g for 5 min at 10 °C and then directly
subjected to HPLC analysis.
Bile samples from rat and dog
Rat bile was continuously collected in fractions for 80 h after intravenous dosing and 96 h after oral
dosing. Dog bile was collected into containers cooled by dry ice for the periods 0–6 and 6–24 h, and
then for 24-h periods up to 96 h after intravenous or oral dosing. At the end of each collection period,
the rat bile was acidified with 1 M hydrochloric acid at a ratio of 10:1 (v/v) and dog bile at a ratio of
9:1 (v/v). For the determination of total radioactivity, 3-μL aliquots of bile were added to 4 mL of
liquid scintillation cocktail and analysed in a Tricarb 2300 TR liquid scintillation counter. For the
recording of metabolic profiles, bile samples were centrifuged at 20800×g for 5 min at 10 °C.
Rat faecal samples
Faeces were collected in parallel to urine and bile over periods of 80 and 96 h, respectively.
Radioactivity was extracted from the samples by suspending aliquots of the faeces in three
equivalents (w/v) of 0.1 M phosphate buffer (pH 7.4) and homogenising with an Ultra Turrax Tube
Drive (IKA, Staufen, Germany). To an aliquot of this suspension, three equivalents (v/w) of a 1:1
(v/v) mixture of methanol and water acidified with 0.1% formic acid were added and the mixture
was homogenised in an Eppendorf ThermoMixer (Hamburg, Germany) for 10 min at 37 °C and
1400 rpm. The resulting suspension was centrifuged at 20800×g for 10 min at 10 °C. The extraction
of radioactivity was repeated twice with acetonitrile as described above. The extracts were combined,
evaporated to dryness in an EZ-2 Evaporator, reconstituted in 500 μL of a 1:1 (v/v) mixture of water
and methanol acidified with 0.1% (v/v) formic acid, centrifuged at 20800×g for 10 min at 10 °C and
subjected to HPLC analysis.
Dog faecal samples
Faeces were quantitatively collected at for 24-h periods up to 96 h post-dose. Radioactivity was
extracted from the samples by suspending aliquots of the faeces in three equivalents (w/v) of
methanol acidified with 0.1% (v/v) formic acid and homogenising with an Ultra Turrax Tube Drive.
The resulting suspension was centrifuged at 20800×g for 10 min at 10 °C. The supernatant was
removed and the pellet extracted twice, each time with 8 mL of a methanol/water (1:1, v/v) mixture.
Accepted Manuscript
The supernatants of all three extraction steps were combined, evaporated to dryness, reconstituted in
500 μL of a 1:1 (v/v) methanol/water mixture acidified with 0.1% (v/v) formic acid, centrifuged at
20800×g for 10 min at 10 °C and subjected to HPLC analysis.
Enzymatic cleavage of glucuronic acid conjugates
Bile and urine samples from rats and dogs were treated with β-glucuronidase to identify potential
glucuronic acid conjugates. For this purpose, appropriate volumes of bile or urine were diluted with
0.1 M phosphate buffer (pH 6.5) to obtain a target radioactivity of about 100 dpm/μL. A 15-μL
aliquot of β-glucuronidase was added and the mixture incubated in an Eppendorf ThermoMixer at
37 °C and 450 rpm for 4 h. The reaction was stopped by the addition of 200 μL of methanol
followed by centrifugation at 20800×g for 10 min at 4 °C prior to HPLC analysis.
Incubation of selexipag with faeces
[14C]Selexipag was incubated at a final concentration of 10 μM with freshly collected faeces from
Sprague-Dawley rats or beagle dogs. Faeces from an untreated rat or dog were weighed and
homogenised in an Ultra Turrax Tube Drive with three equivalents (w/v) of 100 mM phosphate
buffer (pH 7.4). Reactions were initiated by the addition of [14C]selexipag and incubated at 37 °C in
a shaking incubator block (500 rpm). After 24 h, reactions were terminated with 1.5 mL of methanol
and the mixture was homogenised on a Vortex mixer for 2 min. The resulting suspension was
centrifuged at 20800×g for 10 min at 4 °C. The supernatants were evaporated to dryness in an EZ-2
Evaporator, reconstituted in 500 μL of a 1:1 (v/v) mixture of water and methanol, centrifuged at
20800×g for 10 min at 4 °C and subjected to HPLC analysis.
Analytical method for metabolite profiling
Metabolic profiles were recorded using HPLC (LC-20AD; Shimadzu, Reinach, Switzerland) coupled
in-line with a Berthold radioflow detector LB509 (Berthold AG, Regensdorf, Switzerland).
Chromatographic separation of selexipag and its metabolites was achieved on a Gemini C18 column
(5 μm; 250 × 4.6 mm ID) at 40 °C and a flow rate of 1.0 mL/min. The radiodetector utilised a
200-μL liquid cell with a scintillant (Optiflow Safe 2; Berthold AG) flow rate of 3 mL/min. The
mobile phase was 25 mM ammonium formate adjusted to pH 4.1 with formic acid as solvent A and
acetonitrile containing 0.1% formic acid as solvent B. The drug and metabolites were eluted with the
following linear gradient program: equilibration at 20% B; 0 to 5 min, 20 to 35% B; 5 to 15 min, 35
to 40% B; 15 to 40 min, 40 to 60% B; 40 to 47 min, 60 to 75% B; 47 to 51 min, 75% B; 51 to 51.5
min, 75 to 98% B; 51.5 to 53 min, 98% B; 53 to 53.1 min, 98 to 20% B; and 53.1 to 58 min, 20% B.
Data acquisition was done using RadioStar software (version 4.6; Berthold AG).
Accepted Manuscript
Metabolite structure identification in rat bile
Dosing and sample collection
To identify metabolites in rat bile after oral dosing of selexipag, bile samples were additionally
collected from male Sprague-Dawley rats (body weight, approximately 250 g; Japan SLC, Shizuoka,
Japan) using bile-duct-cannulated animals. [14C]Selexipag (three rats) or unlabelled selexipag (four
rats) was intravenously administered at a dose of 1 mg/kg via the jugular vein. The radioactive dose
was 112 μCi/kg for the [14C]selexipag dosing group. After dosing, rats were housed individually in
glass metabolic cages (Metabolica; Sugiyama-Gen, Tokyo, Japan) for separate collection of bile,
urine and faeces. Bile samples were collected into containers on ice up to 3 or 6 h after dosing.
During collection, the bile samples were acidified with 1 M hydrochloric acid to suppress
post-sample degradation of the putative MRE-269 acyl glucuronides.
Chemical cleavage of glucuronic acid
Rat bile samples were treated with alkali to investigate intramolecular acyl migration in glucuronic
acid conjugates. To a 100-μL aliquot of rat bile was added 20 μL of 1 M sodium hydroxide, after
which the mixture was homogenised on a Vortex-mixer and incubated at 37 °C for 30 min. The
reaction was stopped by the addition of 20 μL of 1 M hydrochloric acid, and the mixture
homogenised on a Vortex-mixer. An equivalent volume of acetonitrile was added and the mixture
homogenised on a Vortex-mixer and centrifuged at 16080×g for 5 min at 4 °C prior to HPLC
analysis.
Incubation of MRE-6001 with rat bile
To identify putative MRE-269 acyl glucuronides, a control incubation with freshly collected
Sprague-Dawley rat bile was performed at a single MRE-6001 concentration of 10 μg/mL. To a
500-μL aliquot of bile, a 5-μL aliquot of a 1-mg/mL MRE-6001 stock solution in acetonitrile was
added and the mixture incubated at 37 °C for 30 min. As a control, the reaction was stopped at 0 min.
A small portion of the mixture was retained separately for analysis of acyl migration as described for
bile samples from bile-duct-cannulated rats. The remaining sample was precipitated by the addition
of an equal volume of acetonitrile followed by centrifugation at 16080×g for 5 min at 4 °C. The
supernatants were directly subjected to HPLC analysis.
Analytical method for metabolite identification
The structures of major metabolites in rat bile samples were identified by LC/radiochromatography
and LC-MS/MS analysis. For metabolites for which standards were not available, a structure was
proposed based on MS-MS fragmentation analysis. Radioactivity profiles were recorded using an
Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) coupled in-line with a 515TR
radiochemical detector (PerkinElmer). Chromatographic separation was achieved on an L-column
ODS (5 μm; 150 × 4.6 mm ID) at 40 °C and a flow rate of 1.0 mL/min. The radiodetector utilised a
500-μL liquid cell with a scintillant (Ultima Flo AP; PerkinElmer) flow rate of 2 mL/min. The
Accepted Manuscript
mobile phase was acetonitrile/10 mM ammonium acetate, pH 4.9 (10:90, v/v) as solvent A and
acetonitrile/10 mM ammonium acetate, pH 4.9 (90:10, v/v) as solvent B. The drug and metabolites
were eluted with the following linear gradient program: 0 to 20 min, equilibration at 40% B; 20 to 25
min, 40 to 50% B; 25 to 25.01 min, 50 to 90% B; 25.01 to 28 min, 90% B; 28 to 28.01 min, 90 to
40% B; and 28.01 to 35 min, 40% B. For LC-MS/MS analysis, an Alliance 2795 HPLC system
(Waters, Milford, MA) was used with a Quattro Micro API triple-quadrupole mass spectrometer
with an electrospray ionisation source. Mobile phase A was purified water and mobile phase B was
acetonitrile. The metabolites were eluted with the following linear gradient program: equilibration at
25% B; 0 to 10 min, 25 to 75% B; 10 to 15 min, 75% B; 15 to 15.01 min, 75 to 100% B; 15.01 to 18
min, 100% B; and 18 to 18.01 min, 100 to 25% B. The flow rate was 0.3 mL/min in LC-MS/MS
analysis. The optimal tune parameters for the detection of selexipag and its metabolites were as
follows: capillary, 3 kV; cone 25 V; extractor, 2 V; source, 120 °C; desolvation, 400 °C. The scan
range was set from m/z 125 to 750. The MS data were processed with MassLynx software (version
4.0; Waters).
Results
Metabolism of selexipag in vitro by liver preparations
The in vitro metabolic profile of [14C]selexipag in liver microsomes was compared among rats, dogs,
and monkeys. The relative percentage of the peak area in the radiochromatograms for each
metabolite after a 60-min incubation is presented in Table 3. When all species during all of the time
course experiments were considered, up to 15 different labelled metabolites of [14C]selexipag were
detected. Representative HPLC radiochromatograms of metabolite profiles in the experiment with
liver microsomes are shown in Figure 2. In rat liver microsomes, the main metabolites were
MRE-269, P19, P21 and P25 (Figure 2). In dog liver microsomes, only five metabolites of selexipag
were observed (Table 3). The main metabolite was MRE-269, which accounted for 28% of the
chromatogram radioactivity and 70% of the total metabolite-related material, followed by P10, P19,
P20 and P29. In monkey liver microsomes, 10 metabolites of selexipag were observed. MRE-269
was the main metabolite, accounting for 26.9% of the total chromatogram radioactivity and 31% of
the total metabolite-related material (Table 3). Whereas the conversion of selexipag reached at least
86% with rat and monkey liver microsomes, conversion was slower with dog liver microsomes,
reaching only 40%. Most products observed with selexipag were also observed with [14C]MRE-269.
In all species, MRE-269 was predominantly formed by liver microsomes in the absence of NADPH
(Table 3). The metabolism of MRE-269 in the presence of NADPH was generally slower than that of
selexipag, with a total [14C]MRE-269 conversion of less than 33% in all investigated species
compared with a total [14C]selexipag conversion of up to 92% (Table 3).
A summary of metabolite abundance after 24 h of incubation with hepatocytes is shown in Table
Accepted Manuscript
4, and the metabolite profiles are shown in Figure 3. The metabolic profile of [14C]selexipag on
incubation with rat hepatocytes revealed a total of 10 metabolites (Table 4). Nine of these were also
observed with dog or monkey hepatocytes while two, P7 and P15, were rat-specific. Conversion of
[14C]selexipag was complete after 24 h of incubation. MRE-269 was by far the major product,
accounting for 84% of the total chromatogram radioactivity. [14C]MRE-269 yielded a total of four
metabolites (Table 4). Of these, P12, P13 and P28 were common to [14C]MRE-269 and
[14C]selexipag, whereas P8 was exclusively observed with [14C]MRE-269.
The metabolic profile observed after incubation of [14C]selexipag with dog hepatocytes was
significantly less complex (Table 4), showing only six metabolites, MRE-269, P2, P4, P5, P6 and
P23. MRE-269 was the major product, accounting for 46% of the total chromatogram radioactivity
and 87% of all metabolite-related activity. With [14C]MRE-269, three metabolites, P8, P23 and P24,
were observed, of which only P23 was common to [14C]MRE-269 and [14C]selexipag.
Incubation of [14C]selexipag with cynomolgus monkey hepatocytes yielded a total of 14
metabolites of which seven were monkey-specific (Figure 3). Five similarly abundant major
products, P9, P10, P16, P27 and MRE-269, were observed. [14C]MRE-269 yielded 10 metabolites,
all of which, except P19, were also observed with [14C]selexipag.
In vivo metabolite profiling
After a single intravenous or oral dose of [14C]selexipag to intact rats or dogs, more than 78% of the
radioactivity is excreted in the faeces with a smaller fraction excreted in the urine, indicating bile as
the major excretion route of selexipag (Ichikawa et al., 2018). In the present study, the metabolism
and excretion of selexipag were investigated in bile-duct-cannulated rats and dogs after oral and
intravenous dosing of 14C-labelled drug and parallel sampling of bile, urine and faeces over periods
of 80 (rats) and 96 h (dogs). Equal volumes of artificial bile were re-infused into the duodenum of
the animals to ensure normal bile homeostasis during the course of the study. Blood was collected at
selected times from bile-duct-cannulated dogs while for rats blood was collected from satellite
animals. Metabolic profiles were recorded from all matrices using HPLC coupled to a radiodetector.
Radiochromatograms of representative rat plasma, urine, bile and faecal samples are presented in
Figure 4. The relative abundance of each metabolite as determined from these chromatograms
together with the total radioactivity was used to estimate the amount of each metabolite as a fraction
of the selexipag dose. The results of this analysis are summarised in Table 5.
After oral administration of [14C]selexipag to rats, the major entity in the plasma was the active
metabolite MRE-269, which accounted for 74% of the total plasma radioactivity. Two other
metabolites, P11 and P14, were also observed. Metabolite P11 was identified as the acyl glucuronide
of MRE-269 by co-chromatography with the synthetic standard MRE-6001 (Figure 4B). No
unchanged [14C]selexipag was observed in the plasma samples. Five metabolites, but no unchanged
Accepted Manuscript
selexipag, were observed in urine samples from bile-duct-cannulated rats. P27 and P28 were the
major products after intravenous dosing. P27 was also the major product after oral dosing, with
smaller amounts of P28. None of the other urinary metabolites individually exceeded 1%. Two new
products, P17 and P21, were formed in urine during incubation with β-glucuronidase (Figure 4F).
Whereas P21 obviously originates from the most abundant metabolite, P27, the origin of P17 is less
clear. A total of eight metabolites was observed in rat bile after intravenous dosing of [14C]selexipag.
Among these, P8 was the major metabolite, accounting for 38% of the dose accompanied by
moderate amounts of MRE-269, P9 and P11 and minor amounts of P10, P16, P21 and P23. The four
major products, MRE-269, P8, P9 and P11, were observed after oral dosing. Metabolites P13, P14
and P27 were only detected after oral dosing, pointing to a pre-systemic origin of these products. To
identify potential phase 2 metabolites, rat bile samples were treated with β-glucuronidase. In this
experiment, metabolite P11 was converted into MRE-269 (Figure 4D) as judged from the changes in
the relative peak areas. A total of eight metabolites was observed in rat faecal samples after
intravenous dosing of [14C]selexipag. Apart from [14C]selexipag and its active metabolite MRE-269,
metabolites P4, P5, P10, P15, P16, P20 and P21 were observed. Selexipag, MRE-269, P5 and P20
were present in the faeces in the range of 0.7–1.7% of the dose, whereas all other products were
below 0.4%. After oral administration of [14C]selexipag, a total of 13 metabolites was observed in rat
faeces (Figure 4G). Unchanged [14C]selexipag was the major single entity, representing 7.1% of the
dose and about 40% of the total radioactivity detected in the sample. Among the metabolites,
MRE-269 was the most prominent product, accounting for about 5.5% of the dose, 31% of the
radioactivity recovered from the faeces and 54% of all metabolite-related material. All other
metabolites were much less abundant. Metabolites P4, P10 and P20 were found in faecal samples
irrespective of the route of administration. P5, P15 and P21 were specific for intravenous
administration whereas all other metabolites were observed after oral administration of
[14C]selexipag. About 0.7% of the radioactive dose was detected in faecal samples after intravenous
dosing, indicating a minor degree of direct secretion of [14C]selexipag into the gastrointestinal lumen.
Incubation with rat intestinal microsomes and freshly collected rat faeces yielded a total of six and
ten metabolites, respectively (Figures 5A and 5B), indicating that MRE-269 detected in rat faeces are
the result of metabolism in the gut wall and/or metabolism by the microflora present in the rat
intestinal lumen.
Radiochromatograms of representative dog plasma samples are presented in Figure 6. The
relative distribution of radioactive metabolites in dog bile, urine and faeces after oral administration
of [14C]selexipag and intravenous administration of [14C]selexipag and [14C]MRE-269 to
bile-cannulated dogs is summarised in Table 6. After oral administration of [14C]selexipag, selexipag
and MRE-269 were the major components circulating in dog plasma, together exceeding 75% of the
chromatogram radioactivity at all time points and accompanied by small amounts of metabolite P4
Accepted Manuscript
(Figure 6A). After intravenous administration of [14C]selexipag, selexipag and MRE-269 were again
the major circulating entities, accounting for more than 63% of the detected radioactivity and
accompanied by metabolites P2, P5 and P19 as minor components (Figure 6B). After intravenous
administration of [14C]MRE-269, unchanged drug was the major component in dog plasma over the
whole time course, and was only accompanied by small amounts of metabolite P20 (data not shown).
After oral and intravenous dosing of [14C]selexipag, five and three polar metabolites, respectively,
were observed in urine samples. The active metabolite MRE-269 was absent from these samples and
unchanged selexipag was only present in trace amounts after oral dosing. After intravenous
administration of [14C]MRE-269, six polar metabolites were again observed in urine but no
unchanged MRE-269. No effect of deconjugating enzyme was observed in the incubation of urine
samples with β-glucuronidase (data not shown). After oral and intravenous administration of
[14C]selexipag, unchanged drug was observed in dog bile, accounting for 34% and 42% of the
administered dose, respectively. Five metabolites, P8, P9, P10, P11 and the active metabolite
MRE-269, were also detected in dog bile irrespective of the dosing route. Of these, MRE-269 and P8
were the most prevalent, accounting 25% and 14% of the oral dose and 21% and 8.9% of the
intravenous dose, respectively. After intravenous dosing of [14C]MRE-269, unchanged drug was the
most prevalent component in dog bile over the whole time course, accounting for 44% of the dose.
P8, P9, P10 and P11 were again the major metabolites. The presence of glucuronic acid conjugates
was tested for in selected dog bile samples by incubation with β-glucuronidase. Only P11
disappeared after incubation, identifying it as a glucuronic acid conjugate (data not shown; see
Figure 4B). After oral administration of [14C]selexipag, MRE-269 was the major component in dog
faeces, accounting for 86% of the chromatogram radioactivity. Unchanged selexipag represented
0.6% of the dose and 6% of the drug-related material in the faeces. Six minor metabolites were
detected. The origin of MRE-269 in the faeces samples was explored by incubating [14C]selexipag
with intestinal microsomes or dog faeces. The conversion of [14C]selexipag to MRE-269 was
insignificant on incubation with intestinal microsomes (data not shown) whereas MRE-269 was the
major product on incubation with faeces (Figure 5C). After intravenous administration of
[14C]selexipag, MRE-269 was the major entity, representing 9.6% of the dose and 96% of the
drug-related material in the faeces, and unchanged selexipag was observed as a minor component.
About 0.5% of the radioactive dose was detected in faecal samples after intravenous dosing,
indicating a minor degree of direct secretion of [14C]selexipag into the gastrointestinal lumen. After
intravenous administration of [14C]MRE-269, the vast majority of the drug-related material detected
in the faeces was unchanged drug with small amounts of metabolites P4 and P8.
Identification of the predominant component in rat bile
Metabolites in rat bile samples collected over a 6-h period after intravenous administration of
Accepted Manuscript
[14C]selexipag were characterised by LC/radiochromatography and LC-MS/MS analysis under
different HPLC conditions. BM6 was the major metabolite, and it was accompanied by metabolites
BM1, BM8 and BM9 (Figure 7A). BM6 was identified as the acyl glucuronide of MRE-269 by
co-chromatography with the synthetic standard MRE-6001 and by β-glucuronidase-mediated
deconjugation (data not shown). In order to investigate acyl migration in glucuronic acid conjugates,
rat bile samples were treated with alkali. Under these conditions, metabolites BM5, BM6, BM7 and
BM8 were converted into MRE-269 (Figure 7B) as judged from the changes in the relative peak
heights. To determine the origin of these metabolites, MRE-6001 was incubated with freshly
collected rat bile. The resulting chromatogram is shown in Figure 7C. Incubation of MRE-6001 with
rat bile yielded metabolites BM5, BM7, BM8 and MRE-269 via acyl migration or hydrolysis.
Metabolites BM1 and BM9 were insensitive to alkaline treatment, indicating the absence of the
glucuronic acid moiety. To elucidate the structure of metabolite BM9, bile samples were collected
from bile-duct-cannulated rats over a 3-h period after intravenous administration of unlabelled
selexipag. A non-radioactive BM9 peak with a retention time identical to that of radioactive BM9
was isolated by HPLC and identified by its mass fragmentation pattern. A mass shift of +107 Da
relative to MRE-269 was observed for BM9 (m/z 527), indicating taurine conjugation. The structure
and MS fragment ions of BM9 are shown in Table 2 and Figure 8A. The key fragment ions
generated were m/z 362, 344, 302 and 260, in agreement with the fragmentation pathways of
MRE-269 (Figure 8B).
Discussion
This study reports the comparative disposition and in vitro and in vivo metabolic profiles of
[14C]selexipag and its active metabolite [14C]MRE-269 in rats, dogs, and monkeys.
Eighteen metabolites were observed on incubation of [14C]selexipag with liver microsomes and
hepatocytes. MRE-269 was by far the most prominent product. Most metabolites were common
products of microsomal and hepatocyte incubation, indicating a major role of phase I metabolism
mediated by CYP/flavin monooxygenase (FMO) in the biotransformation of selexipag. Metabolite
patterns of [14C]MRE-269 were similar to those of [14C]selexipag but the conversion of
[14C]MRE-269 was significantly lower. The active metabolite MRE-269 was formed with liver
microsomes even in the absence of NADPH, so that the hydrolysis of [14C]selexipag appears to be
an esterase-catalysed reaction rather than a P450-catalysed one. This is consistent with previous
observations that MRE-269 formation in liver microsomes is suppressed by esterase inhibitors
(Nakamura et al., 2007). Based on the overall comparison of the metabolite patterns of both
compounds generated by incubation with liver microsomes and hepatocytes, two alternative
pathways are likely to operate in parallel yielding the same final products, i.e., either initial
CYP/FMO-catalysed metabolism followed by subsequent hydrolysis, or initial formation of
Accepted Manuscript
MRE-269 followed by phase I metabolism via CYP/FMO.
Some interspecies differences were evident for both compounds in the incubations with liver
microsomes and hepatocytes in terms of both total conversion and metabolic profiles. However,
MRE-269 formation was consistently the main pathway and [14C]MRE-269 conversion was
significantly lower in all three species. Significant species differences in the plasma stability of
[14C]selexipag and non-radiolabelled selexipag have been reported (Ichikawa et al., 2018; Nakamura
et al., 2007). The carboxylesterases responsible for selexipag hydrolysis are present in rodent plasma
but absent from the plasma of non-rodent animals and man (Li et al., 2005).
The active metabolite MRE-269 was found in rat and monkey plasma after oral dosing (Ichikawa
et al., 2018) and was also formed in dog plasma in the present study. The plasma exposure exceeded
that of the parent drug, selexipag, in all species. In rats, MRE-269 was the major entity circulating in
the plasma after oral dosing, and it was accompanied by its acyl glucuronide P11 and metabolite P14.
In dogs, unchanged [14C]selexipag and/or its active metabolite [14C]MRE-269 were by far the major
radiolabelled components circulating in the plasma, irrespective of the route of administration. After
oral and intravenous administration of [14C]selexipag to dogs, one (P4) and three (P2, P5, P19) other
(minor) metabolites, respectively, were detected in the plasma, and after intravenous administration
of [14C]MRE-269 only a single minor metabolite, P20, was observed in the plasma. This information
on the exposure levels of circulating metabolites in rats and dogs will contribute to the quantitative
comparison of exposure to these metabolites between humans and animals.
In the incubation of the metabolite standard MRE-6001 with rat bile, the primary acyl
glucuronide P11 (MRE-6001) was converted into a mixture of the corresponding isomeric 2-O-,
3-O-, and 4-O-acyl glucuronides, consistent with the fact that acyl migration predominates over
hydrolysis under physiological conditions (Akira et al., 1998). 1-O-Acyl glucuronides have been
shown to form covalent adducts with proteins (Volland et al., 1991; Dubois et al., 1993;
Kretz-Rommel et al., 1994; Presle et al., 1996), and there is speculation that the protein adducts are
at least partially responsible for the immunological side effects of carboxylate drugs
(Spahn-Langguth et al., 1992; Zia-Amirhosseini et al., 1995).
Full recovery of drug-related material was demonstrated from urine and faeces in the human
absorption, distribution, metabolism, and excretion study with [14C]selexipag, indicating no retention
of radiolabel in humans (Kaufmann et al., 2012). Drugs given at daily doses below 10 mg have not
been associated with idiosyncratic toxicity (Uetrecht et al., 2001; Uetrecht et al., 2013). In the light
of the low concentrations of acyl glucuronides in human plasma and the low selexipag dose used in
humans (Bruderer et al., 2014; Kaufmann et al., 2015), the risk of immune-mediated idiosyncratic
drug reactions as a consequence of covalent binding in patients treated with selexipag is considered
minimal.
Our metabolite-profiling studies in excreta from bile-duct-cannulated rats demonstrated that
Accepted Manuscript
[14C]selexipag was extensively metabolised prior to excretion. Selexipag was not found in the urine,
and only 0.9% of the administered dose was recovered as unchanged drug from the bile. Four major
metabolites were consistently observed in the bile after intravenous or oral administration of
[14C]selexipag to rats, i.e., the active metabolite MRE-269, its acyl glucuronide P11, and metabolites
P8 and P9, indicating limited pre-systemic metabolism in the rat. Intravenous administration of
[14C]selexipag to rats confirmed P11 as a major product in the bile, identical to BM6 observed after
intravenous administration of [14C]MRE-269. Several isomers of P11 (BM6) were observed, likely
resulting from intramolecular acyl migration (Figure 7A). MRE-269 was also conjugated with
taurine (Table 2). In bile-duct-cannulated dogs, [14C]selexipag was partly metabolised prior to
excretion, mainly to its active metabolite MRE-269. Unchanged [14C]MRE-269 accounted for 54%
of the dose recovered after intravenous administration, also suggesting a moderate extent of
metabolism preceding its excretion. Three other important metabolites were consistently observed in
the bile after administration of either compound to dogs, irrespective of the dosing route, i.e.,
MRE-269 acyl glucuronide P11 and metabolites P8 and P9.
Rat urine contained several polar metabolites of unidentified chemical nature. P27 was identified
as the glucuronic acid conjugate of P21. In dog urine, unchanged selexipag was present only in trace
amounts and MRE-269 was not detected at all. MRE-269 and unchanged selexipag were the major
entities in rat and dog faeces, likely resulting from biliary secretion of MRE-269 itself and
hydrolyzed P11, as well as incomplete oral absorption. Selexipag hydrolysis to MRE-269 was
observed in rat faeces ex vivo and also slowly with rat intestinal microsomes. Both observations
point to the occurrence of selexipag hydrolysis already in the gastro-intestinal lumen rather than
during its passage through the gut wall. Similar to the rat, selexipag was hydrolyzed ex vivo with dog
faeces, and slowly in dog intestinal microsomes. Taken together, these data indicate some
pre-systemic loss of selexipag as a result of hydrolysis in the gut.
In summary, selexipag was hydrolysed to an active metabolite, MRE-269, and was extensively
metabolised via oxidative pathways in rats, dogs, and monkeys. The metabolite profile presents an
interesting combination of elimination pathways, including hydroxylation, conjugation, intestinal
deconjugation, and gut flora metabolism. The formation of MRE-269 was the major pathway in all
species, and MRE-269 conversion was significantly lower than selexipag conversion, irrespective of
species. However, some interspecies differences observed for selexipag in terms of total conversion
as well as of metabolic profiles need to be considered in the interpretation of pharmacological and
toxicological observations.
Accepted Manuscript
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Table 1 Summary of [14C]selexipag or [14C]MRE-269 doses, routes of administration and timing of
biological sample collection in rats, dogs.
Species
Route
Single dose
Number
Matrix
Sampling times
Rat
[14C]selexipag
p.o. a
20 mg/kg
(200 μCi/kg)
2
Plasma
0.5 h
[14C]selexipag
p.o. b
20 mg/kg
(100 μCi/kg)
2
Bile
0–2, 2–4, 4–8, 8–24, 24–32, 32–48, 48–72, 72–96 h
Urine
0–2, 2–4, 4–8, 8–24, 24–32, 32–48, 48–72, 72–96 h
Faeces
0–8, 8–24, 24–32, 32–48, 48–72, 72–96 h
[14C]selexipag
i.v. b
0.9 mg/kg
(100 μCi/kg)
2
Bile
0–2, 2–4, 4–8, 8–24, 24–32, 32–48, 48–72, 72–80 h
Urine
0–4, 4–8, 8–24, 24–32, 32–48, 48–72, 72–80 h
Faeces
0–8, 8–24, 24–32, 32–48, 48–72, 72–80 h
Dog
[14C]selexipag
p.o. b
2 mg/kg
(100 μCi/kg)
2
Plasma
5 min, 1, 2, 4, 8, 24, 48 h
Bile
0–6, 6–24 h, daily until 96 h
Urine
0–6, 6–24 h, daily until 96 h
Faeces
Daily until 96 h
[14C]selexipag
i.v. b
1 mg/kg
(100 μCi/kg)
2
Plasma
5 min, 1, 2, 4, 8, 24, 48 h
Bile
0–6, 6–24 h, daily until 96 h
Urine
0–6, 6–24 h, daily until 96 h
Faeces
Daily until 96 h
[14C]MRE-269
i.v. b
1 mg/kg
(100 μCi/kg)
2
Plasma
5 min, 1, 2, 4, 8, 24, 48 h
Bile
0–6, 6–24 h, daily until 96 h
Urine
0–6, 6–24 h, daily until 96 h
Faeces
Daily until 96 h
a Intact rats
b Bile-duct-cannulated animals
Accepted Manuscript
Table 2 Proposed structures of selexipag metabolites in rats.
Structure
[M + H]+
Fragment ions
-
-
420
378, 362, 344, 302, 260
-
-
527
485, 362, 344, 302, 260
-, not determined.
Accepted Manuscript
Table 3 Distribution of radioactive metabolites after incubation of [14C]selexipag or [14C]MRE-269
with liver microsomes from rat, dog, and monkey a).
Rat
Dog
Monkey
Selexipag
MRE-269
Selexipag
Selexipag
MRE-269
Selexipag
Selexipag
MRE-269
Selexipag
+NADPH
+NADPH
-NADPH
+NADPH
+NADPH
-NADPH
+NADPH
+NADPH
-NADPH
Selexipag [47.5] b)
8.1
55.7
59.9
75.3
14.0
30.4
MRE-269 [45.9]
13.0
67.1
44.4
27.9
85.8
24.7
26.9
71.0
69.6
P4 [37.8]
2.3
P8 [34.8]
2.2
P10 [32.6]
2.9
3.6
12.6
P12 [29.9]
5.1
4.9
12.8
9.2
P13 [29.3]
1.9
2.6
P14 [24.5]
5.8
5.0
P19 [16.0]
10.0
4.4
3.1
2.8
1.7
P20 [14.9]
2.5
P21 [12.4]
21.6
7.7
1.6
2.6
P22 [11.2]
4.0
P23 [10.0]
7.2
4.1
3.1
P24 [9.0]
4.9
P25 [8.3]
12.6.
4.2
4.3
7.0
3.1
P26 [7.0]
2.5
P28 [5.4]
.
7.8
7.1
P29 [4.1]
3.0
3.0
2.7
P32 [32.1]
2.7
P33 [6.5]
3.7
7.6
3.1
a) All values are expressed as a percentage of the total chromatogram radioactivity; b) Retention time
in minutes. Empty cells indicate the absence of a metabolite in the sample.
Accepted Manuscript
Table 4 Distribution of radioactive metabolites after incubation of [14C]selexipag or [14C]MRE-269
with hepatocytes from rat, dog, and monkey a).
Rat
Dog
Monkey
Selexipag
MRE-269
Selexipag
MRE-269
Selexipag
MRE-269
Selexipag [47.5] b)
47.5
MRE-269 [45.9]
83.8
94.6
45.6
92.9
14.0
11.8
P1 [54.3]
2.1
2.0
1.5
P2 [44.5]
0.4
P4 [37.8]
1.0
0.5
P5 [36.4]
1.3
P6 [35.9]
2.8
P7 [35.3]
5.6
P8 [34.8]
1.3
5.2
P9 [33.9]
10.4
7.2
P10 [32.6]
0.7
10.6
7.5
P12 [29.9]
0.8
1.2
2.2
P13 [29.3]
1.9
1.2
3.0
P15 [22.8]
2.0
P16 [21.6]
11.4
9.6
P18 [16.8]
5.4
9.1
P19 [16.0]
7.5
P20 [14.9]
6.5
18.4
P22 [11.2]
4.0
5.2
P23 [10.0]
2.1
1.2
P24 [9.0]
0.8
P25 [8.3]
3.5
4.0
P27 [6.1]
1.6
13.5
14.6
P28 [5.4]
0.7
1.8
2.7
P29 [4.1]
4.1
a) All values are expressed as a percentage of the total chromatogram radioactivity; b)Retention time
in minutes. Empty cells indicate the absence of a metabolite in the sample.
Accepted Manuscript
Table 5 Distribution of radioactive metabolites in bile, urine, and faeces after oral or intravenous
dosing of [14C]selexipag to rats a).
p.o.
i.v.
Bile
Urine
Faeces
Bile
Urine
Faeces
Selexipag [47.5] b)
0.90
7.09
0.73
MRE-269 [45.9]
15.3
5.54
13.0
1.70
P2 [44.5]
0.84
P4 [37.8]
0.78
0.32
P5 [36.4]
1.16
P6 [35.9]
0.21
P8 [34.8]
17.1
38.2
P9 [33.9]
12.4
13.4
P10 [32.6]
1.29
0.30
2.47
0.02
P11 [31.5]
20.7
16.7
P12 [29.9]
0.19
P13 [29.3]
0.82
P14 [24.5]
0.67
0.18
P15 [22.8]
0.27
P16 [21.6]
5.06
4.70
0.08
P18 [16.8]
0.50
P20 [14.9]
0.66
0.83
P21 [12.4]
0.09
0.15
0.20
P22 [11.2]
0.26
P23 [10.0]
0.11
P24 [9.0]
0.10
P25 [8.3]
0.11
P26 [7.0]
0.20
P27 [6.1]
3.49
4.01
0.25
2.31
P28 [5.4]
0.37
1.57
P29 [4.1]
0.11
0.33
0.30
P30 [45.2]
0.15
a) Data are presented as a percentage of the dose, b)Retention time in minutes. Empty cells indicate
the absence of a metabolite in the sample.
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Table 6 Distribution of radioactive metabolites in bile, urine, and faeces after oral or intravenous
dosing of [14C]selexipag or [14C]MRE-269 to dogs a).
Selexipag p.o.
Selexipag i.v.
MRE-269 i.v.
Bile
Urine
Faeces
Bile
Urine
Faeces
Bile
Urine
Faeces
Selexipag [47.5] b)
33.7
0.10
0.59
42.2
0.48
MRE-269 [45.9]
24.5
8.83
20.6
9.55
43.7
8.37
P1 [54.3]
0.15
P4 [37.8]
0.22
0.12
0.83
P6 [35.9]
0.13
P8 [34.8]
13.5
8.94
16.3
1.12
P9 [33.9]
3.76
4.61
6.98
P10 [32.6]
0.61
0.87
2.01
P11 [31.5]
3.76
4.11
12.3
P12 [29.9]
0.04
P13 [29.3]
0.23
P14 [24.5]
0.10
P15 [22.8]
0.11
P16 [21.6]
0.03
P19 [16.0]
0.33
0.26
0.41
0.14
P20 [14.9]
0.11
0.12
P22 [11.2]
0.21
P23 [10.0]
0.17
P24 [9.0]
0.31
0.21
0.06
0.22
P25 [8.3]
0.11
0.13
P26 [7.0]
0.14
P28 [5.4]
0.37
0.34
1.06
P29 [4.1]
0.20
P31 [26.8]
0.08
a) Data are presented as a percentage of the dose, b)Retention time in minutes. Empty cells indicate
the absence of a metabolite in the sample.
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Fig. 1. Chemical structures of [14C]selexipag and [14C]MRE-269. Asterisks indicate the labelled
positions. The radiolabel is uniformly distributed throughout both phenyl rings in both compounds.
Figure 2 Metabolic profile of selexipag after incubation with rat liver microsomes.
Figure 3 Metabolic profile of selexipag after incubation with monkey hepatocytes.
Figure 4 Metabolic profiles of selexipag in rat plasma (A), plasma spiked with synthetic MRE-6001
(B), urine (C), urine after β-glucuronidase treatment (D), bile (E), bile after β-glucuronidase
treatment (F) and faeces (G) after oral administration of [14C]selexipag.
Figure 5 Metabolic profiles of selexipag after incubation with rat intestinal microsomes (A), freshly
collected rat faeces (B) and freshly collected dog faeces (C).
Figure 6 Metabolic profiles of selexipag in dog plasma from 2 h post-dose after oral dosing (A) and
plasma from 5 min post-dose after intravenous dosing (B) of [14C]selexipag.
Figure 7 Biotransformation profiles of [14C]selexipag metabolites in 0–6-h rat bile (A) and
alkaline-treated bile (B), and UV chromatogram following 30 min incubation of MRE-6001 with
freshly collected rat bile (C).
Figure 8 Product-ion mass spectrum of metabolite BM9 at m/z 527, (M + H)+ (A) and mass spectral
fragmentation of MRE-269 (B).
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